The present invention relates to novel methods for the preparation of water-soluble cross-linked conjugates and conjugate complexes as well as to novel water-soluble cross-linked conjugates and conjugate complexes per se. The conjugates and conjugate complexes confer an improved sensitivity in immunochemical assays, in particular when used in lateral flow devices and in methods for determining the presence or absence of small amounts of active components present in liquid samples.
A large research effort has been devoted to devising ways to improving immunochemical assay reliability and sensitivity in e.g. home pregnancy and fertility tests and, consequently, there is a continuous need for new and improved methods for preparing conjugates which exhibit a high degree of sensitivity and specificity when employed in such immunochemical assays. Clearly, the number of xe2x80x9cactivexe2x80x9d components, such as the number of antibodies or antigens, and the number of xe2x80x9cdetectable unitsxe2x80x9d, such as dye molecules, present in the conjugate are of utmost importance when developing new conjugates for use in high-sensitive immunochemical assays.
Various strategies for improving the sensitivity and reliability of immunoassays have been reviewed by L. J. Kricka (1994) Clin. Chem. 40, 347-357.
EP 0 594 772 B1 relates to water-soluble, polymer-based conjugates comprising moieties derived from divinyl sulfone. EP 0 594 772 B1 describes the possibility of enhancing the attachment of molecular species, such as antibodies and antigens, to a water-soluble carrier molecule by taking advantage of the so-called xe2x80x9csalting outxe2x80x9d effect. It turned out, however, that by increasing the salt concentration to about 1 M an irreversible precipitate was formed.
It has now surprisingly been found that by further increasing the concentration of salt in the reaction mixture, a reversible (i.e. a re-dissolvable) precipitate is formed which contains xe2x80x9clargexe2x80x9d water-soluble conjugates which are useful in various immunochemical assays, such as in lateral flow devices.
In a first aspect the present invention relates to a method for the preparation of a water-soluble cross-linked conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one spacer component, moieties of at least one signal component and moieties of at least one primary targeting component, the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component, said method comprising:
a) reacting a water-soluble intermediate conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one spacer component, moieties of at least one signal component, the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component,
via reaction of unreacted reactive moieties derived from the linking component, with at least one primary targeting component in an aqueous solution, the conditions being such that a reversible precipitate is formed;
b) re-dissolving the reversible precipitate comprising the water-soluble cross-linked conjugate in an aqueous medium; and
c) optionally subjecting the water-soluble cross-linked conjugate to a purification step.
In the present context the term xe2x80x9cwater solublexe2x80x9d when used in connection with the cross-linked conjugates means that the conjugates obtained according to the methods disclosed herein should be soluble in an aqueous medium, such as water, at room temperature, i.e. the cross-linked conjugates obtained by the methods disclosed herein should give rise to a solution which is substantially clear and homogenous as judged by visual inspection of the sample.
In a preferred embodiment of the invention the cross-linked conjugates obtained by the methods disclosed herein have a water solubility of at least 0.1, preferably at least 1, such as at least 10, more preferably at least 50, such as at least 100, in particular at least 200 mg dry conjugate per ml water at 25xc2x0 C.
Before going into a detailed discussion with respect to the above-mentioned precipitation step, it should be noted that the water-soluble intermediate conjugate may be prepared by a method comprising:
I) reacting at least one water-soluble carrier component with more than one linking component in an aqueous solution at a pH above 7, so as to form an aqueous solution containing a water-soluble intermediate precursor comprising water-soluble moieties of the carrier component having covalently attached thereto reactive moieties derived from the linking component;
II) optionally subjecting the water-soluble intermediate precursor to a purification step;
III) reacting the optionally purified water-soluble intermediate precursor, via reaction of said reactive moieties, with
i) at least one spacer component in an aqueous solution at a pH above 7, so as to form a second water-soluble intermediate precursor, the conditions being such that only a fraction of the reactive moieties reacts with the spacer component and that a significant amount of unreacted reactive moieties remain,
ii) optionally subjecting the second water-soluble intermediate precursor to a purification step, and
iii) reacting the optionally purified second water-soluble intermediate precursor, via reaction of the spacer component, with at least one signal component in an aqueous solution at a pH above 7, so as to form a water-soluble intermediate conjugate, the conditions being such that most of the signal components react with the spacer moiety, rather than with the linker component, and that a significant amount of unreacted reactive moieties of the linker component remain unreacted ( i.e. only a small fraction of the signal components react with the reactive moieties of the linker components); and
IV) optionally subjecting the water-soluble intermediate conjugate obtained in step III) to a purification step.
The method outlined in general form above in steps I-IV, and the components alluded to therein, is schematically represented in FIG. 4. The Figure is merely to be used for purposes of clarity as it represents anecdotal examples of one embodiment. Therein, the various stages of intermediates and precursors that are part of the method are schematically represented to assist the reader to follow the procedure.
The Water-Soluble Carrier Component
The term xe2x80x9ccarrier componentxe2x80x9d in the context of the present invention is used to denote the xe2x80x9cbackbonexe2x80x9d of the conjugate, i.e. the carrier component functions as a backbone on which various molecules may be attached.
The water-soluble polymers which function as the carrier component in the method for the preparation of conjugates may be chosen from a wide variety of types of polymers, including:
natural and synthetic polysaccharides, as well as derivatives thereof, for example dextrans and dextran derivatives, starches and starch derivatives, cellulose derivatives, amylose and pectin, as well as certain natural gums and derivatives thereof, such as gum arabic and salts of alginic acid;
homopoly(amino acid)s having suitable reactive functionalities, such as polylysines, polyhistidines or polyornithines;
natural and synthetic polypeptides and proteins, such as bovine serum albumin and other mammalian albumins; and
synthetic polymers having nucleophilic functional groups, such as polyvinyl alcohols, polyallyl alcohol, polyethylene glycols and substituted polyacrylates.
Very suitable polymers for the purposes of the invention are polysaccharides and derivatives thereof, for example: dextrans, carboxymethyl-dextrans, hydroxyethyl- and hydroxypropyl-starches, glycogen, agarose derivatives, and hydroxyethyl- and hydroxypropyl-celluloses. As will be apparent from the working examples herein (vide infra), notably dextrans have proved to be particularly suitable polymers in connection with the invention, and they are presently the most preferred carrier components.
As already indicated, it is often desirable, particularly for many of the immunochemical applications of the conjugates, that said conjugates have no, or substantially no, net charge, since the presence of a net positive or negative charge in such cases can lead, inter alia, to undesirable non-specific binding of the conjugates to substances and/or materials other than those of interest. In many cases this condition will, unless charged species are introduced, be fulfilled simply by ensuring that the polymeric carrier component itself possesses no net charge. Thus, a preferred polymeric carrier component for use in the method of the invention is, in its free state, substantially linear and substantially uncharged at a pH in the range of about 4 to about 10, the latter pH interval being the interval of practical relevance for the vast majority of immunochemical procedures, hybridisation procedures and other applications of conjugates. Among various polymers which meet this criterion, are, for example, numerous polysaccharides and polysaccharide derivatives, e.g. dextrans and hydroxyethyl- and hydroxypropylcelluloses.
Depending on the use to which a conjugate is to be put, the conjugates may be based on water-soluble polymeric carrier components having a range of molecular weights. In one embodiment of the invention, the polymeric carrier component may have a peak molecular weight in the range of about 40,000 to about 40,000,000 (prior to reacting said water-soluble polymeric carrier components with linker reagent such as DVS or EPCH, or reacting resulting water-soluble intermediate precursor with a spacer or signal component for the eventual formation of cross-linked conjugate and cross-linked conjugate complexes). Peak molecular weights which are of considerable interest are peak molecular weights in the range of 100,000 to 10,000,000, such as in the range from 500,000 to 8,000,000, preferably in the range from 500,000 to 4,000,000, e.g. in the range from 500,000 to 2,000,000. Peak molecular weights of particular interest, notably in the case of dextrans as polymeric carrier components, are peak molecular weights of about 500,000, about 1,000,000, about 1,500,000, about 2,000,000, 2,500,000, about 3,000,000, about 3,500,000 and about 4,000,000.
More particularly, dextrans in the molecular weight ranges of 20,000 to 2,000,000 are preferred as starting carrier components. Most particularly, 20,000 Da dextrans are preferred for, but not restricted to, conjugates and/or complexes using streptavidin as the primary or secondary target. Furthermore, 500,000 Da dextrans are preferred for, but not restricted to, conjugates and/or complexes using certain dyes, enzymes, and with certain specific binding molecules as the primary or secondary target. Moreover, 2,000,000 Da dextrans are preferred for, but not restricted to, certain other dyes.
The term xe2x80x9cpeak molecular weightxe2x80x9d as employed in the present specification and claims in connection with the carrier components denotes the molecular weight of greatest abundance, i.e. that molecular weight, among a distribution of molecular weights, which is possessed by the greatest number of molecules in a given sample or batch of the polymer. It is quite normal to characterise numerous types of polymers in this manner, owing to the difficulty (particularly for the highest molecular weights) of obtaining or preparing polymer fractions of very narrow molecular weight distribution. In the case of numerous commercially available carrier components which are of interest in the context of the invention, for example dextrans, the manufacturer or distributor will be able to provide reliable peak molecular weight data (determined, for example, by gel-permeation chromatography) which can provide a basis for the selection of the proper fraction of the polymeric carrier component. It should be mentioned here that peak molecular weight values (when used in connection with the carrier component) cited in the present specification and claims refer to the peak molecular weight of the free polymer in question, and take no account of, for example, the possible formation of cross-linked polymer units, e.g. as a result of cross-linking of two or more polymer molecules by reaction with a linking component such as DVS or EPCH during a method for the preparation of a conjugate; such cross-linked units will, on average, have higher molecular weights than the individual free polymer molecules from which they are formed.
Formation of the Water-Soluble Intermediate Precursor
In the present context the xe2x80x9cterm linking componentxe2x80x9d is intended to cover bi-functional molecules capable of establishing covalent links between otherxe2x80x94typically largerxe2x80x94molecules. Examples of linking components suitable for the method according to the invention are e.g. molecules comprising a bi-functional reactivity such as glutaraldehyde, carbodiimides, N,Nxe2x80x2-phenylenedimaleimide, N-succinimidyl 3-(2-pyridylthio)propionate, p-benzoquinone, bis oxiranes, divinyl sulfone (DVS) and epoxide derivatives, such as epoxides of the general formula I: 
wherein R1 is hydrogen or C1-4-alkyl, n is an integer in the range from 1-4, i.e. 1, 2, 3 or 4, and X is a leaving group such as tosyl, mesyl, or halogen such as fluorine, chlorine, bromine, or iodine, preferably chlorine.
In the present context the term xe2x80x9cC1-4-alkylxe2x80x9d designates a straight or branched saturated hydrocarbon group having from 1 to 4 carbon atoms, such as methyl ethyl, n-propyl, n-butyl, isopropyl, isobutyl, etc.
As will be apparent from the working examples provided herein a very promising epoxide-derived linking component is epichlorohydrin (EPCH), i.e. a compound of the general formula I above, wherein R1 is hydrogen, n is 1 and the leaving group X is chlorine.
Preferably, the linking component should be stable in an aqueous environment and, accordingly, the linking component EPCH constitutes together with the linking component DVS the presently most preferred linking components for use in the method of the invention.
The first step, i.e. step I), wherein the water-soluble intermediate precursor is formed, is conveniently carried out in an aqueous solution at a pH above 7, such as above 8.5, in particular above 9, such as above 10, for example at a pH around 10, 10.5, 11 or 11.5. In its most general form, the reaction may take place at a temperature in the range of 0-60xc2x0 C., although a temperature in the range of 20-25xc2x0 C. will often be quite suitable, as illustrated, for example, for carrier components such as dextrans, in the working examples given herein. In a preferred embodiment of the invention, the pH at which the reaction takes place is generally within the range of about 10-11.5, which is a pH range in which the reactive functionalities on most types of carrier components are reactive towards the presently preferred linking components DVS and EPCH.
As far as the concentration of the carrier component in the aqueous solution is concerned, it will generally be within the range of 0.1-20% w/v, and often in the range of 0.5-10% w/v, such as in the range of 0.5-5% w/v, in particular in the range from 0.5-2% w/v, such as about 0.5% w/v, about 1% w/v, about 1.5% w/v or about 2% w/v. The concentration of linking component in the aqueous solution will generally be in the range of 0.1-35% v/v, depending on the actual linking component employed. The concentration of the presently preferred linking components, i.e. DVS and EPCH, in the aqueous solution is typically in the range from 0.1-15% v/v in the case of DVS, and often in the range of 1-10% v/v. In the case of EPCH the concentration is typically in the range from 1-30% v/v, and often in the range from 3-20% v/v. In case the where the reagent for the preparation of the linking component is a solid, it is contemplated that the concentration will generally be in the range of 0.1-10% w/v.
It is difficult to give general guidelines concerning the period of time for which the reaction of linking component with the carrier component in the aqueous solution should be allowed to proceed, since these will vary rather considerably, depending on, e.g., the temperature and pH at which the reaction occurs, the concentration of the carrier component and the concentration of linking component in the reaction mixture, the nature and/or molecular weight of the carrier component and the nature of the linking component, and the extent to which cross-linking of the carrier (e.g. by reaction with DVS) may proceed before there is a risk, for example, of gelling or precipitation taking place.
The reaction time in question will, however, normally be within the range of 5 minutes to 10 hours. As will be apparent from the working examples provided herein, the reaction time required when DVS is used as the linking component is typically in the range from 5 to 120 minutes, such as in the range from 15 to 60 minutes, e.g. about 30 minutes, whereas activation of the carrier component with EPCH in general requires a longer reaction time, typically in the range from 1 to 10 hours, such as in the range from 3 to 7. hours, e.g. about 5 hours.
As already discussed, the carrier component in the water-soluble intermediate precursor has covalently attached thereto one or more moieties derived from a bifunctional linking component, each of which moieties is attached via a covalent linkage formed between one of the two functional groups of the bifunctional linking component and a reactive functionality on the carrier component. As will be understood, the remaining functional group of the bi-functional linking component will be free (xe2x80x9cdanglingxe2x80x9d) and, consequently, be capable of reacting with e.g. primary targeting components, spacer components and/or signal components under suitable conditions (vide infra).
The xe2x80x9cloadxe2x80x9d, i.e. the number of linking groups attached to the carrier component [in step I)], will typically be in the range from about 1 to about 5,000 moles of linking components per gram of carrier component, such as in any of the following sub-ranges (expressed as xcexcmoles linking component per gram of carrier component): about 1 to about 50; about 50 to about 300; about 300 to about 1,000; or about 1,000 to about 5,000. The number of linking groups attached to the carrier component may be determined by titration methods known per se, e.g. by the thiosulphate titration method described in Porath et al. (1975) J. Chromatogr. 103, 49. As is apparent from examples provided herein, the typical xe2x80x9cloadxe2x80x9d of the linker, expressed in xcexcmoles of linker per gram of carrier ranges from approximately 300 to more than 2000. Thus, in preferred embodiments of this aspect of the invention, the range of about 1 to about 5,000 moles of linking components per gram of carrier component should particularly be between 200 and 3000, preferably between 500 and 2500.
The optional purification step (step II) may, for example, involve a process such as dialysis (for the removal of excess reagent or other species of low molecular weight) or some chromatographic technique which will be suitable for the purpose, such as gel-filtration. It should be understood, however, that the above-mentioned purification methods are only mentioned as examples and the skilled person will be able to select the most appropriate purification method in each individual case, which may depend on the actual conditions employed in the coupling step, the actual ingredients used in the coupling step as well as available equipment at the site of production.
Carrier components (which, as explained above, constitute the xe2x80x9cbackbonexe2x80x9d of the conjugates) which are suitable for use in the method of the invention are preferably initially non-cross-linked and are of essentially zero charge at pH values which are of relevance within the fields of application of the invention.
Owing to the nature of the coupling chemistry employed in the method according to the invention, i.e. the establishment, on the carrier component, of covalently bound reactive moieties deriving from bi-functional molecules, such as DVS and EPCH, will generally require that a reactive functionality, preferably a nucleophilic functionality, is present on the carrier component. Suitable carrier components will then be, for example, polymeric carrier components with functional groups such as: xe2x80x94Oxe2x88x92 (e.g. deprotonated phenolic hydroxy groups, such as deprotonated aromatic hydroxy groups in tyrosine residues of polypeptides or proteins), xe2x80x94Sxe2x88x92 (e.g. deprotonated thiol groups on aromatic rings or aliphatic groups, such as deprotonated thiol groups in cysteine residues of polypeptides or proteins), xe2x80x94OH (e.g. aliphatic hydroxy groups on sugar rings, such as glucose or other monosaccharide rings in oligo- or polysaccharides; or alcoholic hydroxy groups in polyols, such as polyvinyl alcohol; or hydroxy groups in certain amino acid residues of polypeptides or proteins, such as serine or threonine residues), xe2x80x94SH (e.g. thiol groups in cysteine residues of polypeptides or proteins), primary amino groups (e.g. in lysine or omithine residues of polypeptides or proteins; or in amino-substituted sugar rings in certain polysaccharides or derivatives thereof, such as chitosan) or secondary amino groups (e.g. in histidine residues of polypeptides or proteins). As will be understood by the skilled person, the question of whether the functional groups mentioned above will be in a protonated or de-protonated state will, of course, depend on the selected reaction conditions, such as the pH of the reaction mixture.
For similar reasons, the functional group in question on targeting components and spacer components (vide infra) in the context of the invention will also normally be a nucleophilic functionality, such as a nucleophilic functionality of one of the above-described types.
Formation of the Second Water-Soluble Intermediate Precursor
In step IIIi) of the method of the invention the spacer component is, via reaction with the linking component, covalently attached to the water-soluble intermediate precursor, thereby forming a second water-soluble intermediate precursor.
As indicated above, the xe2x80x9cspacer componentxe2x80x9d is covalently attached, via the linking group, to the carrier component. Thus, the term xe2x80x9cspacer componentxe2x80x9d when used in the present context is intended to mean a protein or a polypeptide which has a plurality of sites available for covalent attachment of signal components, such as dyes (vide infra).
One purpose for the incorporation of a spacer component, and particularly for a spacer having a plurality of sites available for covalent attachment of signal components, is that this method provides for a suitable means of increasing the number of signal components which can be attached to the conjugate (i.e. the xe2x80x9cloadxe2x80x9d of the signal component in the water-soluble intermediate conjugate, vide ante), and thereby increasing the sensitivity of such conjugates when employed in various assays, e.g. immunochemical assays and in the lateral flow devices described herein (vide infra). It should be understood that in an embodiment wherein the coupling of a signal component (such as a dye molecule) is done directly to the linking component (and not through a spacer component) implies that (at least in principle) only one signal molecule is attached per molecule of linking component present in the conjugate.
In several embodiments of the preparation of the second water-soluble precursor, the number of moles of spacer per mole of starting dextran (the xe2x80x9cloadxe2x80x9d of the spacer) ranges from 1 to 500, particularly from 2 to 100, most frequently from 5 to 75. Also, as explained in details in Example 3A herein, the second water-soluble intermediate (and hence the efficiency of the reaction carried out in step IIIi)) may be characterised by e.g. the number (moles) of spacer component attached per mole carrier component.
As stated earlier, only a fraction of the reactive moieties of the linking component of the water-soluble intermediate reacts with the spacer component. Depending on the spacer component and on the linker component, after reacting the spacer component, from 1 to 99% of the unreacted reactive moieties of the linker component, preferably 20-99%, particularly 30-99%, such as ranging from 40 to 99% and notably 50 to 99% remain unreacted. That is to say that, in one embodiment, under certain conditions, from 1 to 49% of the unreacted linker moieties reacted with the spacer component.
Preferably, the spacer component is a protein such as BSA, ovalbumin, globulin, etc. or a polypeptide such as homopolypeptides, e.g. polylysines, polyhistidines, polyornithines, etc. However, as will be clear to a person skilled in the art, the choice of spacer component will depend on the employed signal component (e.g. the actual dye employed in a particular conjugate) as well as the employed linking component.
The molecular weight of the spacer component, e.g. a protein, is preferably at least 10,000 Da, preferably in the range of 10,000-2,000,000, such as in the range of 20,000-500,000. As the one of the features of the introduced spacer components is to multiply the number of available positions for introduction of the signal components, it is furthermore preferred that the number of available functional groups for attachment of signal components is at least 5 per molecule of spacer component, preferably 10-1,000, in particular 10-500.
Alternatively, the spacer component can be a polysaccharide or polynucleic acid. Chemical modifications of these polymers may be required prior to the preparation of the water-soluble intermediate conjugate.
As stated earlier, owing to the nature of the coupling chemistry on the spacer component, (to both the linker component in the formation of the second water-soluble intermediate precursor, or later to a signal component in the formation of the water-soluble-intermediate conjugate, vide infra), a reactive functionality, such as a nucleophilic functionality, is present on the spacer component. Suitable spacer components will then be, for example, those with nucleophilic functional groups such as: xe2x80x94Oxe2x88x92 (e.g. deprotonated phenolic hydroxy groups, such as deprotonated aromatic hydroxy groups in tyrosine residues of polypeptides or proteins), xe2x80x94Sxe2x88x92 (e.g. deprotonated thiol groups on aromatic rings or aliphatic groups, such as deprotonated thiol groups in cysteine residues of polypeptides or proteins), xe2x80x94OH (e.g. aliphatic hydroxy groups present in certain amino acid residues of polypeptides or proteins, such as serine or threonine residues), xe2x80x94SH (e.g. thiol groups in cysteine residues of polypeptides or proteins), primary amino groups (e.g. in lysine or omithine residues of polypeptides or proteins) or secondary amino groups (e.g. in histidine residues of polypeptides or proteins). As will be understood by the skilled person, the question of whether the functional groups mentioned above will be in a protonated or de-protonated state will, of course, depend on the selected reaction conditions, such as the pH of the reaction mixture.
Step IIIi) of the method of the invention, wherein the second water-soluble intermediate precursor is formed, is conveniently carried out in aqueous solution at a pH above 7, such as above 8, in particular above 9, such as above 10, for example at a pH in the interval of from 10 to 11, e.g. of from 10 to 10.5. It will normally be quite sufficient to carry out the reaction at a temperature in the range from 0-60xc2x0 C., the optimal temperature being dependent on, inter alia, the actual pH employed. In most cases, especially when the reaction is carried out at a pH above 9, and in particular when the reaction is carried out at a pH above 10, a temperature in the range of 20-40xc2x0 C., e.g. around 30xc2x0 C., will often be quite suitable. With respect to the reaction time, it should be understood that several parameters will influence the reaction time required. Thus, depending on the employed pH, the reaction temperature, the concentration of peptide or polypeptide spacer component and the concentration of water-soluble intermediate precursor, the reaction time may vary within wide limits. It is contemplated, however, that a suitable reaction time will generally be in the range of from 1 hour to 48 hours and, as will be understood from the examples provided herein, the present inventors have found that by using the specified set of reaction conditions disclosed in Examples 3A and 3B, a reaction time in the range of from 10 to 30 hours, e.g. in the range from 15 to 25 hours such as about 18 hours, is quite suitable.
As stated earlier, only a fraction of the unreacted reactive moieties of the linker component of the water-soluble intermediate react with the spacer component. That is to say that the second water-soluble intermediate still possesses a significant amount of unreacted reactive moieties.
The obtained second water-soluble intermediate precursor may be purified by the methods already discussed in connection with the purification step II), i.e. in connection with the purification of the water-soluble intermediate precursor. As will be evident from the examples provided herein, a suitable method for purifying the obtained second water-soluble intermediate precursor is gel-filtration.
Formation of the Water-Soluble Intermediate Conjugate
In step IIIiii), the signal component is, via reaction with the spacer component, covalently attached to the second water-soluble intermediate precursor, thereby forming a water-soluble intermediate conjugate.
When used herein, the term xe2x80x9csignal componentxe2x80x9d is intended to cover such components which are directly physically detectable or which are precursors for such physically detectable components. In other words, the signal component should function as a label or a marker which can be readily measured by some physical technique known in art, e.g. by means of optical methods, such as spectrophotometry, fluorescence, luminescence, phosphorescence or other methods such as those described in e.g. xe2x80x9cInstrumental Methods of Chemical Analysisxe2x80x9d G. W. Ewing, 5th Ed., McGraw-Hill Book Company, New York, 1988. Alternatively, the signal component mayxe2x80x94as indicated abovexe2x80x94be a precursor for a such physically detectable component. A typical example of a such precursor is an enzyme which upon action on a suitable substrate is capable of generating species, preferably coloured species, which can be detected by one or more of the physical methods mentioned above.
In light of the discussion given above, it will be clear to the skilled person that the signal component may be selected from substances such as dyes; fluorescent, luminescent, phosphorescent and other light-emitting substances; metal-chelating substances, including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA) and desferrioxamine B; substances labelled with a radioactive isotope; substances labelled with a heavy atom; and mixtures thereof.
To give some further examples, fluorescent substances may be selected from, e.g., fluorescein (suitably as fluorescein isothiocyanate, FITC), fluoresceinamine, 1-naphthol, 2-naphthol, eosin, erythrosin, morin, o-phenylenediamine, rhodamine and 8-anilino-1-naphthalenesulfonic acid. Radioactive isotopes of relevance may be selected, for example, among isotopes of hydrogen (i.e. tritium, 3H), carbon (such as 14C), phosphorus (such as 32P), sulfur (such as 35S), iodine (such as 131I), bismuth (such as 212Bi), yttrium (such as 90Y), technetium (such as 99mTc), palladium (such as 109Pd) and samarium (such as 153Sm). Heavy atoms of relevance may be selected, for example, among Mn, Fe, Co, Ni, Cu, Zn, Ga, In, Ag, Au, Hg, I, Bi, Y, La, Ce, Eu and Gd. Gold (Au) is a particularly useful heavy atom in many cases.
Signal components which are considered of particular interest are the dyes. In the present context the term xe2x80x9cdyexe2x80x9d is intended to mean any spectrophotometrically detectable dye molecule or derivative thereof. Preferred dyes to be incorporated in the conjugates prepared by the methods according to the invention are derived from visual dyes, phosphorescent dyes, fluorescent dyes, laser dyes, infrared dyes and lanthanide chelates. Dyes which are particular interesting are visual dyes, including soluble visual dyes, such as solvent dyes, pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species such as fluorescein, rhodamine and derivatives thereof (such as sulphorhodamine, rhodamine-hydride and rhodamine hydrazide), as well as oxazine dyes, cyanine dyes and azol dyes. Specific examples of suitable dyes are, for example, Texas Red hydrazine, Congo Red, Trypan Blue, Lissamine Blue, Remazol Black, Remazol Brilliant Red, Rhodamine B Isothiocyanate, Cy5-Osu mono functional reactive dye, Reactive Orange 16, Uniblue A, etc.
The above-mentioned dyes, which are useful as signal components for the purposes of the present invention, are all well-known in the art and it will be clear to the skilled person that other dyes can be used as signal components for the purposes of the present invention. Other examples of dyes to be used as signal components are e.g. such dyes as mentioned in xe2x80x9cDyeing and Chemical Technology of Textile Fibersxe2x80x9d, Trotman, 34th Ed., C. Griffin and Co., London and xe2x80x9cThe Chemistry of Synthetic Dyesxe2x80x9d, Vankataramon (Ed.), Academic Press, New York, 1979, the disclosures of which are incorporated herein by reference.
Preferably, the signal component should be capable of reacting with a protein, such as BSA and/or, for alternative embodiments described below, capable of reacting with an unreacted reactive moiety of a linker component). Furthermore, the signal component, upon reacting or binding to the spacer, should preferably not confer any undesirable properties of the resulting water-soluble intermediate conjugate, i.e. the signal component should preferably not promote any uncontrollable non-specific binding nor inhibit the activity of the targeting components (e.g. antibodies) bound to the conjugate. Furthermore, the signal component should preferably not reduce the water solubility of the conjugate significantly.
Depending on the size of the starting dextran, the type of signal component used, and particularly depending on the xe2x80x9cloadxe2x80x9d of the spacer, the xe2x80x9cloadxe2x80x9d of the signal component will obviously vary. As stated earlier, each spacer is able to accommodate several signal components. In preferred embodiments, the number of signal components per spacer component ranges from 1 to 100, expressed in moles of each component. Particularly interesting are the embodiments where the molar ratio ranges from 2 to 80, notably 2 to 75.
As stated earlier, only a small fraction of the reactive moieties of the linking component of the second water-soluble intermediate reacts with the signal component in the formation of the water-soluble intermediate conjugate. Depending on the signal component, the spacer component, and on the linker component, after reacting the signal component, and relative to the amount of unreacted reactive linking component available in the second water-soluble intermediate precursor), from 50 to 100% of the unreacted reactive moieties of the linker component, preferably 60-100%, particularly 70-100%, such as ranging from 80-100% and notably 90-100% remain unreacted (N.B. as compared to the second water-soluble intermediate precursor).
Depending on the particular dye, the conjugate prepared by the method of the invention absorbs or emits photons in the visible range, in the UV range or in the near infrared range, preferably in the visible range. Use of a visual dye such as rhodamine will cause the conjugate of the invention to absorb photons in the visible region (e.g. blue), resulting in the transmission of the complementary wavelength of colour (e.g. red) to an observer. Alternatively, the use of a fluorescent dye will (when radiated) cause the conjugate of the invention to emit photons at a specific wavelength due to the return of electrons to the ground state.
Step IIIiii) of the method of the invention, wherein the water-soluble intermediate conjugate is formed, is conveniently carried out in aqueous solution at a pH above 7, such as above 8, in particular in a range from about 8 to about 11, such as in the range from about 8.5 to 10.5. Depending on the actual signal component employed, the aqueous reaction mixture may contain from 0-60% v/v of an organic co-solvent. Thus, in order to dissolve rather hydrophobic signal component (such as certain dye molecules) it may be necessary to add various amounts of a water-miscible organic co-solvent, such as dimethylsulfoxide (DMSO), ethanol, dimethylformamide (DMF), etc. to the aqueous reaction mixture in order to ensure a sufficient solubility of the employed signal component. In order to avoid denaturation of the previously coupled spacer components (which are typically polypeptides or proteins) the concentration of organic co-solvent in the reaction mixture should preferably be as low as possible.
In a similar way as described above in connection with the steps concerning the formation of the water-soluble intermediate precursor and the formation of the second water-soluble intermediate precursor, it will also in this step be sufficient to carry out the reaction at a temperature in the range from 0-60xc2x0 C., such as in the range from 20-40xc2x0 C., e.g. about 30xc2x0 C.
As will be apparent from the working examples provided herein, the reaction time may be varied within wide limits. Thus the reaction time may depend on, e.g., the xe2x80x9cloadxe2x80x9d of spacer component on the carrier component as well as the usual reaction parameters, such as pH, the temperature, and the nature and concentration of the reactants. In general, however, the reaction time will be in the range of from 1 to 48 hours. Preferably, the reaction time should be as low as possible, i.e. in the range of from 1 to 24 hours, in particular in the range of from 1 to 12 hours, such as in the range of from 1 to 5 hours.
In a similar way as described above, the obtained intermediate conjugate may be purified by a number of different techniques known to the skilled person. Further, the obtained intermediate conjugate may be isolated in a solid form by means of, for example, freeze drying or evaporation of the solvent. In case of the latter, the evaporation is typically carried out under reduced pressure, e.g. by means of a (evacuated) desiccator. The obtained intermediate conjugate may be characterised in various ways. If, for example, the employed signal component is a visual dye, its absorbance can be read and the intermediate conjugate (and hence the efficiency of the coupling step IIIiii)) may, for example, be expressed as the number of Extinction Units (EU) present in the intermediate conjugate per mg of spacer component such as described in Example 4A, herein. The skilled person will, of course, be able to characterise the obtained intermediate conjugate in a number of other ways.
It should be noted that in the method of the invention discussed so far, the spacer component is coupled, via the linking group, to the carrier component after which the signal component is attached to the spacer component. Thus, the spacer component is already attached to the carrier component (via the linking group) when the signal component (such as a dye) is coupled to the spacer component.
Alternatives to the Formation of the Water-Soluble Intermediate Conjugate
As stated earlier, and as will be understood from the examples provided herein, the method is also suitable for the preparation of water-soluble cross-linked conjugates wherein the signal component is covalently attached to the linking component, which in turn is attached to the carrier component, i.e. no protein or polypeptide spacer component is incorporated in the conjugate (vide infra).
In such cases the signal component may, of course, in addition to the signal components mentioned above, also be selected from substances such as proteins, including ferritin, phycoerythrins, phycocyanins and phycobilins; enzymes, including horseradish peroxidase, alkaline phosphatase, glucose oxidases, galactosidases and ureases; and mixtures thereof.
As will be obvious to the skilled person, the signal component may also be covalently attached to the spacer component prior to coupling of the spacer component to the carrier component (via the linking group).
In one preferred embodiment, under certain conditions, only a fraction of the reactive moieties of the linking component of the water-soluble intermediate reacts with the signal component. Depending on the linker component, after reacting the signal component, from 1 to 99% of the unreacted reactive moieties of the linker component, preferably 1-89%, particularly 1-69%, such as ranging from 1 to 59% and notably 1 to 49% remain unreacted. That is to say that in preferred embodiments, from 50 to 99% of the reactive moieties reacted with the signal component.
Accordingly, in another interesting embodiment, the water-soluble intermediate conjugate may be prepared by a method comprising:
I) reacting at least one water-soluble carrier component with more than one linking component in an aqueous solution at a pH above 7, so as to form an aqueous solution containing a water-soluble intermediate precursor comprising water-soluble moieties of the carrier component having covalently attached thereto reactive moieties derived from the linking component;
II) optionally subjecting the water-soluble intermediate precursor to a purification step;
III) reacting the optionally purified water-soluble intermediate precursor, via reaction of said reactive moieties, with at least one spacer component to which at least one signal component has been covalently attached, in an aqueous solution at a pH above 7, so as to form a water-soluble intermediate conjugate, the conditions being such that only a fraction of the reactive moieties reacts with the spacer component to which at least one signal component has been covalently attached; and
IV) optionally subjecting the water-soluble intermediate conjugate obtained in step II) to a purification step.
The purification/isolation process may be by methods already discussed in connection with the optional purification of the water-soluble intermediate precursor.
Formation of the Water-Soluble Cross-Linked Conjugate
Turning now to a more detailed discussion of the precipitation step, it will be understood by the skilled person the xe2x80x9ckey stepxe2x80x9d in the method of the invention is step a), wherein the primary targeting component is attached to the intermediate conjugate, the reaction conditions being such that a reversible precipitate is formed.
In the present context, the term xe2x80x9creversible precipitatexe2x80x9d is intended to mean that the precipitate formed is capable of being re-dissolved upon dilution with water at 25xc2x0 C. The term xe2x80x9cprimary targeting componentxe2x80x9d, as used herein, is intended to designate molecules, especially molecules of biological origin, which are capable of selectively binding to, or selectively reacting with, a complementary molecule or a complementary structural region of a material of biological origin. Examples of relevant primary targeting components are, for example: antigens; haptens; monoclonal and polyclonal antibodies; gene probes; natural and synthetic oligo- and polynucleotides; natural and synthetic mono-oligo- and polysaccharides; lectins; avidin; streptavidin; biotin; growth factors; hormones; receptor molecules; protein A and protein G; and mixtures thereof.
Examples of primary targeting components which are considered to be of particular interest for the purpose of the present invention are e.g. anti human Chorionic Gonadotropin (anti hCG), Rabbit anti human CRP, streptavidin, avidin, anti HIV, anti hepatitis C, anti Chlamydia, anti herpes, anti thyroid stimulating hormone (anti TSH), anti Listeria, and anti salmonella.
Examples of relevant primary targeting components, which are hormones, are steroid hormones (e.g. estrogen, progesterone or cortisone), amino acid hormones (e.g. thyroxine) and peptide and protein hormones (e.g. vasopressin, bombesin, gastrin or insulin). EP 0 594 772 B1 mentions at page 12, lines 20-38, that the effectiveness, when attaching molecular species (such as antibodies) to a carrier (such as dextran) may be increased by taking advantage of the so-called xe2x80x9csalting outxe2x80x9d effect, and it is stated that a suitable concentration will be a concentration corresponding to an ionic strength in the range of from 0.5-5. The examples disclosed in EP 0 594 772 B1 demonstrate that a positive effect with respect to the amount of various species coupled to the carrier was in fact obtained if the salt concentration was increased to a certain level. However, when the salt concentration was increased to about 1 M an irreversible precipitate was formed.
As already mentioned, the present inventors have now surprisingly found that by further increasing the concentration of lyotropic salt in the reaction mixture, a reversible precipitate is formed which contains xe2x80x9clargexe2x80x9d conjugates which: i) are believed to be extensively a cross-linked, ii) are water-soluble, and iii) have a high sensitivity (due to a xe2x80x9chighxe2x80x9d load of targeting component and/or a xe2x80x9chighxe2x80x9d load of signal component) when used in various assays, such as in the lateral flow devices disclosed herein (vide infra). The advantages of the water-soluble cross-linked conjugates, which may be obtained by the methods described herein, will be discussed in details below.
Without being bound by a specific theory it is presently believed that the presence of salt in the reaction mixture causes the activity coefficient of the intermediate conjugate to increase, thereby decreasing the solubility of the intermediate conjugate. In a similar way, the activity coefficient of the primary targeting component (e.g. an antibody) will increase, thereby decreasing the solubility of the primary targeting component. Thus, one hypothesis may be that when the intermediate conjugate as well as the primary targeting component are precipitated (probably together with some co-precipitated water) the two reactants are brought very close together and thereby increasing the probability that a chemical reaction takes place, i.e. increasing the probability that the primary targeting component reacts with the previously unreacted reactive moieties of the linking component. It should be emphasised, however, that the exact mechanism has not presently been solved in detail and, in principle, the extensive cross-linking/attachment of primary targeting component may occur in solution after which the cross-linked conjugate precipitates, or the reaction may take place as the precipitation occurs, or the reaction may occur after the precipitation has taken place (as discussed above). It should be emphasised, however, that irrespective of the actual mechanism by which the cross-linking/attachment of primary targeting component takes place, it can be concluded that the reversible precipitation obtained when using the methods according to the present invention does notxe2x80x94contrary to the teaching disclosed in EP 0 594 772 B1xe2x80x94lead to conjugates having such properties that irreversible precipitation occurs.
Without being limited to a specific theory, it is presently believed that the cross-links established in connection with the precipitation step is constituted, at least to some extent, by the bi-functional linking components, i.e. the first reactive moiety of the linking component is covalently attached to a reactive functionality on a first moiety of a carrier component and the second reactive moiety of the linking component is covalently attached to a reactive functionality on a second moiety of a carrier component. As an illustrative example, the establishment of a cross-link between to dextran carrier components using DVS as linking component may, for example, be as follows:
Dextran-Oxe2x80x94CH2xe2x80x94CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2xe2x80x94O-Dextran. 
It is contemplated, however, that cross-linking of the individual carrier components in the precipitation step may be facilitated by the primary targeting component and, accordingly, a cross-link between e.g. two dextran carrier components where e.g. DVS is used as the linking component, may, for example, have the following structure:
Dextran-Oxe2x80x94CH2xe2x80x94CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2xe2x80x94xe2x80x9cprimary targeting componentxe2x80x9dxe2x80x94CH2xe2x80x94CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2O-Dextran 
or
Dextran-Oxe2x80x94CH2CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94CH2-xe2x80x9cprimary targeting componentxe2x80x9dxe2x80x94O-Dextran. 
Probably, more than one primary targeting component is incorporated in some of the cross-links and it is contemplated that the primary targeting component may react with a third or even with a fourth linking component thereby establishing cross-links between more than two moieties of carrier components. In fact, the primary targeting component may, at least in principle, react with as many linking components as it possesses reactive sites.
The degree of cross-linking is believed to be directly related to the amount of unreacted reactive moieties of the linker component available to react during the xe2x80x9csalting-outxe2x80x9d process. The amount of unreacted reactive moieties remaining after the spacer coupling (formation of the second water-soluble intermediate precursor) in preferred embodiments ranges from 50 to 99%, and whereupon subsequent coupling of the signal component (formation of the water-soluble intermediate conjugate), the amount of unreacted reactive moieties, in preferred embodiments, remains unchanged in ranging from 50 to 99%, the amount of reactive moieties available for cross-linking and thus potential for a high degree of cross-linking is great. Clearly, the more extensive the cross-linking (potentially via one or more linkers, between two dextrans, through a spacer or through a primary target) the greater the molecular weight of the conjugate. Obviously, the degree of cross-linking is also related to the method employed for the reversible precipitation step.
The precipitation step a) in the method of the invention is conveniently carried out in an aqueous solution at a pH in the range of 6-11, preferably in the range of 6-10, e.g. in the range of 8-10, in particular in the range of 8-9.
With respect to the reaction time and reaction temperature these parameter may be varied depending on the concentration and/or the nature of the reactants employed. It has been found by the present inventors, however, that a suitable reaction temperature is typically in the range from 2-30xc2x0 C., such as in the range from 4-16xc2x0 C., preferably in the range from 4-10xc2x0 C., in particular at 4-6xc2x0 C.
The reaction time may be varied between 1 to 36 hours, usually between 6 to 24 hours, e.g. between 15 to 21 hours, such as about 18 hours.
In interesting embodiments of the invention, the initial molar ratio in the solution (i.e. before any precipitation occurs) between the intermediate conjugate and the primary targeting component is in the range from 1:1 to 1:50, such as in the range from 1:1 to 1:25, e.g. in the range from 1:1 to 1:10, preferably in the range from 1:1 to 1:5, in particular in the range from 1:2.5 to 1:5.
The reversible precipitation is preferably performed by salting-out which is conveniently obtained by means of adding lyotropic salts to the reaction mixture. Examples of suitable lyotropic salts are, for example, sulphates, phosphates, citrates or tartrates of lithium, sodium, calcium, potassium or ammonium, or mixtures thereof. Further examples of lyotropic salts are given in xe2x80x9cPurification Tools for Monoclonal antibodiesxe2x80x9d, Gagnon, P., Validated Biosystems, 1996, hereby incorporated by reference.
In presently preferred embodiments of the salting-out process, the lyotropic salts calcium phosphate and ammonium sulfate have been particularly effective.
The concentration of the lyotropic salt should be sufficient to ensure that the reversible precipitation process yields a cross-linked conjugate. The concentration of the salt required to effectuate the desirable effect is dependent on the nature of both the cation and anion of the lyotropic salt. As stated earlier, salt concentrations of up to 1 M resulted in the formation of an irreversible precipitate (EP 0 594 772 B1) and did not effectuate the desirable effect. However, salt concentrations greater than 1 M, such as ranging from 1.25 to 3 M, yield the desired cross-linked conjugates. As stated the salt concentration for a particular reversible precipitation will vary according to the choice of the salt used. Moreover, the salt concentration for a particular reversible precipitation will vary according to the load and nature of each of the components. The load and choice of the linker component, the spacer component and signal component will affect the precise salt concentration (of a particular choice of salt). In preferred embodiments, the lyotropic salt concentrations range from 1.25 to 2.75 M, such as at least 1.25 M, or at least 1.5 M, or at least 1.75 M, or at least 2 M, or at least 2.2.5 M, or at least 2.5 M, or at least 2.75 M.
The lyotropic salt should be present in a concentration which is sufficient to ensure that a reversible precipitate is formed, i.e. the concentration of lyotropic salt, namely calcium phosphate or ammonium sulfate, is preferably in the range 1.25 to 2.75 preferably in the range 1.75 to 2.50 M.
Although, as explained above, the salt precipitation step very efficiently couples primary targeting components, any remaining free xe2x80x9cdanglingxe2x80x9d groups derived from the linking component may be deactivated by adding deactivating species of low molecular weight to the aqueous solution containing the reversible precipitate. Examples of suitable deactivating species may be, for example, ethanolamine, mercaptoethanol, or certain amino acids such as cysteine, glycine, alanine or valine.
Following the reversible salt precipitation step, the (reversibly) precipitated conjugates are re-dissolved in an aqueous medium, preferably water. The conjugates obtained according to the methods disclosed herein should be well-soluble in an aqueous medium, such as water, at room temperature and should preferably have a water solubility of at least 0.1, preferably at least 1, such as at least 10, more preferably at least 50, such as at least 100, in particular at least 200 mg of dry conjugate per ml of water at 25xc2x0 C.
Obviously, and as will be clear to the skilled person, the obtained water-soluble cross-linked conjugate may be further purified and/or isolated in a solid form by means of, for example, freeze drying. The purification/isolation process may be by methods already discussed in connection with the optional purification of the water-soluble intermediate precursor.
Although the precipitation step preferably is carried out by means of addition of lyotropic salt to the reaction mixture it is envisaged that the step, wherein cross-linking/attachment of primary targeting component occurs, may be carried out by means of other techniques than salt precipitation. Thus, one example of an alternative to the above-mentioned salt precipitation is to carry out the reaction in a frozen aqueous solution, i.e. at a temperature in the range from about xe2x88x9220xc2x0 C. to 0xc2x0 C. In such frozen solutions small xe2x80x9cpocketsxe2x80x9d of water will occur, wherein the reactants will be present in a very high concentration, thereby increasing the probability that a chemical reaction takes place, i.e. increasing the probability that the primary targeting component reacts with the previously unreacted reactive moieties of the linking component. Still other examples of methods which are contemplated to be useful in the method of the invention include, for example, solvent precipitation, i.e. addition of water-miscible organic solvents to the aqueous reaction mixture; polymer precipitation, i.e. addition of one or more inert polymers to the aqueous reaction mixture; various concentration techniques, such as evaporation, preferably under reduced pressure, etc. The common feature for all the above-mentioned techniques is the enhancement of the proximity of the reactants, thereby increasing the probability that the primary targeting component reacts with the previously unreacted reactive moieties of the linking component.
In embodiments wherein the water-soluble cross-linked conjugate is purified by freeze-drying and wherein any remaining unreacted reactive moieties of the linker component have not been deactivated (using a deactivating species), the level of cross-linking may be augmented by the freeze-drying process. Given that the above stated hypothesis regarding the mechanism by which the cross-linked conjugate is formed is founded on the proximity of the reactants, freeze-drying may simulate, to some extent, in one or more aspects, the salting-out process. Thus, in this embodiment, the level of cross-linking and hence the mean molecular weight may increase compared to that prior the purification process. Conversely, in another embodiment, the unreacted reactive moieties of the linker component are deactivated by said methods prior to the optional purification process.
As already indicated, the main importance of the water-soluble cross-linked conjugates prepared by the methods disclosed herein is presently seen in connection with their use in lateral flow devices, which will be discussed in details below. Therefore, the present inventors have provided a suitable lateral flow device assay which enables the skilled person to select effective and preferred water-soluble cross-linked conjugates prepared by the methods according to the invention. Thus, Example 7A discloses a test for the sensitivity of water-soluble cross-linked conjugates prepared by the methods disclosed herein. It should be noted that the test, when used exactly as described herein, is only suitable for conjugates, wherein the signal component is a visual dye and the primary targeting component is Rabbit anti Human CRP. However, the skilled person will know how to expand the test to encompass other signal components and, more important, other primary targeting components.
As will be acknowledged by the skilled person and as will be apparent from the working examples provided herein, the methods disclosed herein does not necessarily produce xe2x80x9cone single typexe2x80x9d of conjugate but rather conjugates having a certain molecular weight distribution. From the above-mentioned test it is possible to assess whether the obtained water-soluble cross-linked conjugate is found suitable for the purpose or if further purification/fractionation is desirable. It has been found by the present inventors that in a very interesting embodiment of the invention, the water-soluble cross-linked conjugates obtained in the precipitation step a) is further purified/fractionated by means of gel-filtration. Thus, water-soluble cross-linked conjugates which are very suitable for the use in, for example, lateral flow device systems are such conjugates which, after being re-dissolved in an aqueous medium, are eluted in the void volume when subjected to gel-filtration using, for example, the gel material Sephacryl(trademark) HR S-500 or Sephacryl(trademark) HR S-1000 (using the conditions specified in the working examples disclosed herein).
As indicated previously, the water-soluble cross-linked conjugates are xe2x80x9clargexe2x80x9d compared to known conjugates. As will be understood by the skilled person, and as mentioned above, the conjugates prepared by the methods disclosed herein will not give rise to a conjugate of a single uniform weight, but rather the obtained conjugates will have a certain molecular weight distribution. Several possible methods, which will be known to the skilled person, may be employed in the determination of different kinds of the average molecular weight of such heterogeneous conjugates. It is envisaged, however, that very suitable methods are, for example, analytical ultracentrifugation and, in particular, light scattering techniques. Thus, in interesting embodiments of the present invention the conjugates obtained by the methods disclosed herein have a mass average molecular weight of at least 106, at least 2xc3x97106, at least 3xc3x97106, at least 4xc3x97106, at least 5xc3x97106, at least 6xc3x97106, at least 7xc3x97106, at least 8xc3x97106, at least 9xc3x97106, at least 107, at least 2xc3x97107, at least 3xc3x97107, at least 4xc3x97107, at least 5xc3x97107, at least 6xc3x97107, at least 7xc3x97107, at least 8xc3x97107, at least 9xc3x97107, at least 108, at least 2xc3x97108, at least 3xc3x97108, at least 4xc3x97108, at least 5xc3x97108, at least 6xc3x97108, at least 7xc3x97108, at least 8xc3x97108, at least 9xc3x97108, at least 109, at least 2xc3x97109, at least 3xc3x97109, at least 4xc3x97109, at least 5xc3x97109, at least 6xc3x97109, at least 7xc3x97109, at least 8xc3x97109, at least 9xc3x97109, at least 1010, at least 2xc3x971010, at least 3xc3x971010, at least 4xc3x971010, at least 5xc3x971010, at least 6xc3x971010, at least 7xc3x971010, at least 8xc3x971010, at least 9xc3x971010, at least 1011, at least 2xc3x971011, at least 3xc3x971011, at least 4xc3x971011, at least 5xc3x971011, at least 6xc3x971011, at least 7xc3x971011, at least 8xc3x971011, at least 9xc3x971011, at least 1012, at least 2xc3x971012, at least 3xc3x971012, at least 4xc3x971012, at least 5xc3x971012, at least 6xc3x971012, at least 7xc3x971012, at least 8xc3x971012, at least 9xc3x971012, or at least 1013 g/mol. Although it is preferred that the conjugates are as large as possible it should be understood that the conjugates should preferably not be larger than the pore size of the solid support material (e.g. nitrocellulose) used in the lateral flow devices as the conjugates should be able to flow in said pores. In a particular interesting embodiment of the present invention the conjugates obtained by the methods disclosed herein have a mass average molecular weight in the range from about 106 to about 1010 Da, preferably in range 106 to about 108 Da such as in the range from about 106 to about 108 Da.
When using the mass average molecular weight the individual conjugates are weighted according to their mass fractions, m/m, in the sample. Thus, in the present context, the term xe2x80x9cmass average molecular weightxe2x80x9d is defined with reference to the formula II below:
 less than M greater than =(1/m)xcexa3imi Mi xe2x80x83xe2x80x83(II) 
wherein  less than M greater than  is the mass average molecular weight, m is the total mass of the sample (i.e. the total mass of the conjugates), and mi is the total mass of molecules (i.e. conjugates) having a molecular weight of Mi.
The gel-filtration profiles (FIGS. 3a-3f) clearly show that the molecular weights of the conjugates prepared in high ionic strength (3a, c, e) are higher than the conjugates prepared in low ionic strength (3b, d, f). Thus an important feature of the present invention lies in that the conjugates prepared by the method of invention are notably different than those prepared to water-soluble polymer-based conjugates prepared by the method described in EP 0 594 772 B1. Structural differences (the degree and nature of the cross-linking) deriving from the method of invention, act in part, along with molecular weight differences and other features to confer activity not previously described for water-soluble polymer-based conjugates.
As mentioned previously in connection with the definition of the term xe2x80x9csignal componentxe2x80x9d the methods disclosed herein are also suitable for the preparation of water-soluble cross-linked conjugates, wherein no spacer component is present, i.e. the signal component, such as an enzyme or a dye molecule, is directly attached, via the linking component, to the carrier component (as described in Alternatives to the Formation of the Water-Soluble Intermediate Conjugate).
Thus, in another aspect the present invention relates to a method for the preparation of a water-soluble cross-linked conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one signal component and moieties of at least one primary targeting component, the signal component being covalently attached, via the linking component, to the carrier component, said method comprising:
a) reacting a water-soluble intermediate conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one signal component, the signal component being covalently attached, via the linking component, to the carrier component,
via reaction of unreacted reactive moieties derived from the linking component, with at least one primary targeting component in an aqueous solution, the conditions being such that a reversible precipitate is formed;
b) re-dissolving the reversible precipitate comprising the water-soluble cross-linked conjugate in an aqueous medium; and
c) optionally subjecting the water-soluble cross-linked conjugate to a purification step.
In a similar way, the water-soluble intermediate conjugate, used for the preparation of the water-soluble cross-linked conjugate as described above, may be prepared by a method comprising:
I) reacting at least one water-soluble carrier component with more than one linking component in an aqueous solution at a pH above 7, so as to form an aqueous solution containing a water-soluble intermediate precursor comprising water-soluble moieties of the carrier component having covalently attached thereto reactive moieties derived from the linking component;
II) optionally subjecting the water-soluble intermediate precursor to a purification step;
III) reacting the optionally purified water-soluble intermediate precursor, via reaction of said reactive moieties, with at least one signal component in an aqueous solution at a pH above 7, so as to form a water-soluble intermediate conjugate, the conditions being such that only a fraction of the reactive moieties reacts with the signal component; and
IV) optionally subjecting the water-soluble intermediate conjugate obtained in step II) to a purification step.
As it appears, the formation of the water-soluble intermediate conjugate in step III) is the step which differs from the previous discussed methods for preparation of the water-soluble intermediate conjugate. In general, step III) above may be carried out under very similar conditions as described previously for the attachment of signal components to the spacer components. Thus, step III) of the method disclosed above, wherein the water-soluble intermediate conjugate is formed, is conveniently carried out in aqueous solution at a pH above 7, such as in the range from about 8 to 12, preferably in the range from about 9 to 12, in particular in the range from 10 to 12 or in the range from 11 to 12. Depending on the actual signal component employed, the aqueous reaction mixture may contain from 0-60% v/v of an organic co-solvent. Thus, in order to dissolve rather hydrophobic signal component (such as certain dye molecules) it may be necessary to add various amounts of a water-miscible organic co-solvent, such as dimethylsulfoxide (DMSO), ethanol, dimethylformamide (DMF), etc. to the aqueous reaction mixture in order to ensure a sufficient solubility of the employed signal component. It will usually be sufficient to carry out the reaction at a temperature in the range from 0-60xc2x0 C., such as in the range from 15-40xc2x0 C., e.g. in the range from 20-25xc2x0 C.
In general the reaction time will be in the range of from 1 to 48 hours. Preferably, however, the reaction time should be as low as possible, i.e. in the range of from 1 to 24 hours, in particular in the range of from 1 to 12 hours, such as in the range of from 1 to 5 hours.
In a particularly preferred embodiment, the use of a dextran with a peak molecular weight of 500,000, with the use of the linking component DVS within activation degree of 20-30%, the use of the spacer component BSA, the use of the signal component Rhodamine B Isothiocyanate and the use of the either the primary targeting components streptavidin or a monoclonal or polyclonal antibody are the components present in the key reversible precipitation step.
As discussed previously the xe2x80x9ckey stepxe2x80x9d in the methods described herein is step a) wherein the primary targeting component is attached to the intermediate conjugate, the reaction being such that a reversible precipitate is formed. Usually, the most expensive reagent to be used for the preparation of the conjugates described herein is the primary targeting component (such as an antibody or an antigen) and, at the same time, the reversible salt precipitation step is one of the most time-consuming steps in the preparation of the conjugates. Furthermore, as the primary targeting will vary depending on the actual target component to be detected a very interesting aspect of the present invention relates to a test kit comprising the water-soluble intermediate conjugate (preferably in the form of a solid) provided with instructions for carrying out the reversible salt precipitation step, the subsequent re-dissolving of the reversible precipitate and the final purification of the thereby formed water-soluble cross-linked conjugate (e.g. by means of gel-filtration). Various modifications of the kit, such as including sets of primary targeting components which are often used in diagnosis/analysis within, for example, the food industry or at hospitals, are also within the scope of the present invention. The kit may, of course, also be provided with instructions for the use of the prepared conjugates in a lateral flow device as described herein.
Formation of the Water-Soluble Cross-Linked Conjugate Complex
In another interesting aspect, the present invention relates to a method for the preparation of a water-soluble cross-linked conjugate complex comprising a conjugate prepared according to any of the methods disclosed herein, a ligand and a secondary targeting component, the ligand being covalently bound to the secondary targeting component, and the ligand being bound to the primary targeting component of the conjugate by means of non-covalent bonds, said method comprising:
I) preparing a water-soluble conjugate according to the methods disclosed herein;
II) reacting the optionally purified water-soluble cross-linked conjugate with a ligand, said ligand being covalently bound to a secondary targeting component, in an aqueous solution;
III) terminating the reaction; and
IV) optionally subjection the water-soluble cross-linked conjugate complex to a purification step.
In the present context the term xe2x80x9csecondary targeting componentxe2x80x9d designates molecules, especially molecules of biological origin, capable of selectively binding to, or selectively reacting with, a complementary molecule or a complementary structural region of a material of biological origin. Thus, the secondary targeting component may be selected from the same class of molecules as mentioned above in connection with the definition of the xe2x80x9cterm primary targeting componentxe2x80x9d, i.e. examples of interesting secondary targeting components are, for example: antigens; haptens; monoclonal and polyclonal antibodies; gene probes; natural and synthetic oligo- and polynucleotides; natural and synthetic mono- oligo- and polysaccharides; lectins; avidin; streptavidin; biotin; growth factors; hormones; receptor molecules; protein A and protein G; and mixtures thereof. In a particular preferred embodiment of the invention the secondary targeting component is anti hCG.
When used herein, the term xe2x80x9cligandxe2x80x9d is intended to mean a molecule having a high affinity for the actual employed primary targeting component, thereby securing a thermodynamically stable non-covalent bond between the ligand and the primary targeting component present in the water-soluble cross-linked conjugate. Thus, in a preferred embodiment of the invention the ligand/primary targeting component are chosen so that the association constant between the ligand and the primary targeting component of the conjugate is at least 106, preferably at least 108, such as at least 1010, more preferably at least 1011, such as at least 1012, in particular at least 1013, such as at least 1014, e.g. at least 1015 I/mol.
As indicated above, the choice of ligand will, of course, be dependent upon the actual primary targeting component employed. Specific examples of suitable ligands are, for example, biotin, anti dinitrophenol or anti dioxygenin, in particular biotin.
In a very interesting embodiment of the invention the ligand/primary targeting component employed is the xe2x80x9cbiotin/streptavidin systemxe2x80x9d or the xe2x80x9cbiotin/avidin systemxe2x80x9d, i.e. the ligand is biotin and the primary targeting component is streptavidin or avidin. As mentioned above, the ligand employed should be covalently bound to a secondary targeting component and, as will be known to the skilled person, biotinylated compounds, such as biotinylated antibodies, are readily available as they can be prepared, for example, as described in Kendall et al. Journal of Immunological Methods (1993), 56, 329-339. Thus, by preparing the water-soluble cross-linked conjugates by the methods disclosed herein, using streptavidin or avidin as the primary targeting component, one would obtain a useful xe2x80x9ctemplatexe2x80x9d onto which any biotinylated secondary targeting component of interest may be attached. It should be understood, however, that any xe2x80x9chapten/antibody systemsxe2x80x9d may be useful as ligand/primary targeting component provided that the association constant between the employed antibody and the employed hapten fulfils the requirements set forth above.
The reaction step II) mentioned above is usually carried out at room temperature after which the reaction may be terminated [step III)] for example by altering the pH of the reaction mixture and/or by addition of excess free ligand, such as biotin.
As will be understood by the skilled person, the water-soluble cross linked conjugate complexes may be purified by the same methods as mentioned previously in connection with the purification of the water-soluble cross-linked conjugates. In addition hereto, it should be noted that the conjugate complexes may, of course, be isolated in a solid form in a similar way as discussed previously in connection with the conjugates.
As the water-soluble cross-linked conjugates and the water-soluble cross-linked conjugate complexes represent a novel class of compounds another aspect of the present invention relates to a water-soluble cross-linked conjugate comprising moieties of at least one carrier component, moieties of more than one linking component, moieties of at least one spacer component, moieties of at least one signal component and moieties of at least one primary targeting component, the signal component being covalently attached to the spacer component and the spacer component being covalently attached, via the linking component, to the carrier component, wherein
the signal component is selected from the group consisting of dyes, proteins (including ferritin, phycoerythrins, phycocyanins and phycobilins), enzymes (including horseradish peroxidase , alkaline phosphatase, glucose oxidases, galactosidases and ureases), fluorescent, luminescent, phosphorescent and other light-emitting substances, metal-chelating substances (including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA) and desferrioxamine B), substances labelled with a radioactive isotope, substances labelled with a heavy atom, and mixtures thereof;
and the spacer component is selected from the group consisting of proteins and polypeptides.
A still further aspect of the present invention relates to a water-soluble cross-linked conjugate complex comprising a water-soluble cross-linked conjugate as defined herein, a ligand and a secondary targeting component, the ligand being covalently bound to the secondary targeting component, and the ligand being bound to the primary targeting component of the conjugate by means of non-covalent bonds.
As will be understood, details and particulars concerning the properties and constituents of the water-soluble cross-linked conjugates as well as the water-soluble cross-linked conjugate complexes of the invention will be the same as, or analogous to, the details and particulars concerning the properties and constituents of the water-soluble cross-linked conjugates as well as the water-soluble cross-linked conjugate complexes discussed in connection with the method aspects above. This means that wherever appropriate, the statements made in connection with the method aspects apply mutatis mutandis to the conjugates and conjugate complexes as such.
Devices and Uses of Conjugates and Complexes
The present invention also relates to a lateral flow device for determining the presence or absence of at least one target component in a liquid sample, said lateral flow device comprising:
I) a test strip comprising an application part, a deposit part and a detection part and being arranged in such a way that the liquid sample can flow from the application part through the deposit part to the detection part;
II) a dry deposit, located in the deposit part of the test strip, of at least one conjugate as defined herein, or a dry deposit of at least one conjugate complex as defined herein, or a mixture thereof; and
III) at least one targeting component capable of selectively binding to, or selectively reacting with, one or more target components present in the liquid sample, the targeting component being immobilised on the detection part of the test strip.
In another interesting aspect, the present invention relates to a method for determining the presence or absence of at least one target component in a liquid sample, said method comprising:
I) adding the liquid sample to the application part of the lateral flow device as defined herein;
II) optionally adding a washing buffer to the application part of the lateral flow device;
III) allowing sufficient time for the applied liquid, and where appropriate the washing buffer, to flow from the application part through the deposit part to the detection part;
IV) detecting the presence or absence of a signal in the detection part.
The conjugate and/or the conjugate complex of the invention is supported (as a dry deposit) on the deposit part of the test strip in such a manner that when wetted, the conjugate and/or the conjugate complex is capable of being transported (in a dissolved state) by capillary forces to the detection part of the test strip.
The targeting component which is supported on the detection part of the test strip is supported in a manner such that the targeting component remains immobile and, consequently, cannot be transported by means of capillary forces. Thus, the targeting component may be supported on the test strip, e.g. by means of adsorption, covalent coupling, etc. Procedures for immobilising targeting components, such as antibodies and antigens, on a support material are generally known in the art.
In one embodiment of the invention, the so-called xe2x80x9csandwichxe2x80x9d technique, in all its variations as is known by the person skilled in the art, is employed for the test analysis.
As will be understood by the skilled person the so-called xe2x80x9capplicationxe2x80x9d part of the test strip, i.e. the part of the test strip which is to be wetted by the sample containing the target component (i.e. the analyte) to be detected, may be identical to the deposit part. Thus, in an interesting embodiment of the invention the sample containing the target component to be detected is applied directly to the part of the test strip comprising the conjugate and/or the conjugate complex.
The test strip is one which is capable of absorbing the target component from the sample applied, and which, when wetted provides for a flow of target component by capillary attraction from the application part through the deposit part (thereby dissolving the dry deposit of conjugate and/or conjugate complex which is then bound to, and transported with, the target component) to the detection part.
The employed strip is made of a material which is capable of supporting the conjugates and/or the conjugate complexes of the invention as well as targeting components such as e.g. antibodies and/or antigens. Examples of suitable materials from which the test strip can be made are e.g. glass fibre, cellulose, nylon, cross-linked dextran, various chromatographic papers, cellulose esters such as nitrocellulose, etc. Presently, the most preferred material is nitrocellulose.
Although referred to as a xe2x80x9cstripxe2x80x9d, wherein the various parts are arranged in the same plane in a manner such that the liquid comprising the target component can flow by capillary attraction from the application part, through the deposit part, to the detection part, the support material may, of course, have any shape or form as long as the requirements with respect to the various parts and flowability are fulfilled.
The liquid comprising the target component to be detected will most often (but not necessarily) be of biological origin such as a blood sample, a serum sample, a plasma sample, a urine sample, a semen sample, or mixtures thereof.
The lateral flow device described herein is capable of detecting small amounts of a variety of target components such as antigens; haptens; monoclonal and polyclonal antibodies; gene probes; natural and synthetic oligo- and polynucleotides; natural and synthetic mono- oligo- and polysaccharides; growth factors; hormones; receptor molecules; as well as mixtures thereof. Specific examples of target components are, for example, hCG, Rabbit human CRP, HIV, hepatitis C, Chlamydia, herpes, thyroid stimulating hormone (TSH), Listeria, Salmonella, and mixtures thereof.
In a particular preferred embodiment of the invention the signal component of the employed water-soluble cross-linked conjugate and/or conjugate complex is a dye which may be directly detectable by the naked eye. Consequently, when such conjugates/conjugate complexes are employed in the lateral flow device disclosed herein it will be possible to visually determine the presence or absence of target component in the applied liquid sample. However, another interesting embodiment of the invention comprises the use of conjugates/conjugate complexes as described herein, wherein the signal component is such a signal component that when applied in the lateral flow device disclosed herein, the signal may be detectable by the naked eye after addition of a reagent to the detection part.
As discussed previously, the conjugates of the present invention are significantly xe2x80x9clargerxe2x80x9d compared to the conjugates disclosed in the prior art. Although it may be difficult to established the exact structure of the conjugates according to the invention, it is presently believed that extensive cross-linking has taken place which in turn is responsible for the size (and thereby the mass average molecular weight) of the conjugates. As will be apparent from the working examples discloses herein, it appears to be a general rule that the higher molecular weight, the better performance (i.e. the higher sensitivity) is obtained when tested in the xe2x80x9cStandard Lateral Flow Performance testxe2x80x9d described in Example 7A, herein.
It should be noted that the lateral flow device disclosed in EP 0 291 194 A1 is an example of a suitable lateral flow device, wherein the water-soluble cross-linked conjugates and/or conjugate complexes of the present invention may be incorporated.
In still further aspects, the present invention also relates to the use a water-soluble cross-linked conjugate, as defined herein, and to the use a water-soluble cross-linked conjugate complex, as defined herein, in immunochemical assay techniques, including enzymatic immunoassays (EIA) such as ELISA, radioimmunoassays (RIA), nephelometric and turbidimeric immunoassays, immunohistochemical procedures, cytochemical procedures, flow cytometry, in situ hybridisation techniques, membrane hybridisation techniques, including Southern and Northern blotting, biosensors, lateral flow devices, or methods based on lectin/carbohydrate interactions.